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PubMed1977

Levels of oxidized and reduced pyridine nucleotides in dormant spores and during growth, sporulation, and spore germination of Bacillus megaterium.

Dormant spores of Bacillus megaterium contained no detectable reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) despite significant levels of the oxidized forms of these nucleotides (NAD and NADP). During the first minutes of spore germination there was rapid accumulation of NADH and NADPH. However, this accumulation followed the fall in optical density that is characteristic of the initiation of spore germination. Accumulation of NADH and NADPH early in germination was not blocked by fluoride or cyanide, and it occurred even when germination was carried out in the absence of an exogenous source of reducing power. In addition to pyridine nucleotide reduction, de novo synthesis also began early in germination as the pyridine nucleotide levels increased to those found in growing cells. Midlog-phase cells grown in several different media had 20 to 35 times as much total pyridine nucleotide as did dormant spores. However, as growth and sporulation proceeded, the NADH plus NAD level fell four- to fivefold whereas the NADPH plus NADP level fell by a lesser amount. From min 10 of spore germination until midway through sporulation the value for the ratio of NADH/NAD is about 0.1 (0.03 to 0.18) while the ratio of NADPH/ANDP is about 1.4 (0.3 to 2.4). Comparison of these ratios in log-phase versus stationary phase (sporulation) growth in all three growth media tested did not reveal any common pattern of changes.

Bacillus megaterium↗
PubMed1993

Molecular cloning and characterization of the Bacillus subtilis spore photoproduct lyase (spl) gene, which is involved in repair of UV radiation-induced DNA damage during spore germination.

Upon UV irradiation, Bacillus subtilis spore DNA accumulates the novel thymine dimer 5-thyminyl-5,6-dihydrothymine. Spores can repair this "spore photoproduct" (SP) upon germination either by the uvr-mediated general excision repair pathway or by the SP-specific spl pathway, which involves in situ monomerization of SP to two thymines by an enzyme named SP lyase. Mutants lacking both repair pathways produce spores that are extremely sensitive to UV. For cloning DNA that can repair a mutation in the spl pathway called spl-1, a library of EcoRI fragments of chromosomal DNA from B. subtilis 168 was constructed in integrative plasmid pJH101 and introduced by transformation into a mutant B. subtilis strain that carries both the uvrA42 and spl-1 mutations, and transformants whose spores exhibited UV resistance were selected by UV irradiation. With a combination of genetic and physical mapping techniques, the DNA responsible for the restoration of UV resistance was shown to be present on a 2.3-kb EcoRI-HindIII fragment that was mapped to a new locus in the metC-pyrD region of the B. subtilis chromosome immediately downstream from the pstI gene. The spl coding sequence was localized on the cloned fragment by analysis of in vitro-generated deletions and by nucleotide sequencing. The spl nucleotide sequence contains an open reading frame capable of encoding a 40-kDa polypeptide that shows regional amino acid sequence homology to DNA photolyases from a number of bacteria and fungi.

Amino Acid Sequence↗
PubMed1995

Molecular dissection of mutations in the Bacillus subtilis spore photoproduct lyase gene which affect repair of spore DNA damage caused by UV radiation.

In response to UV irradiation, Bacillus subtilis spore DNA accumulates the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, or spore photoproduct (SP). SP is broken down into monomers during spore germination by the product of the spl gene which has been proposed to encode the enzyme SP lyase. The wild-type spl gene was cloned by complementation of a mutation designated spl-1; the putative spl gene product is a 40-kDa protein whose deduced amino acid sequence contains regions homologous to DNA photolyases. During phenotypic characterization of spl subclones using transformation crosses between the cloned wild-type spl gene and an spl-1 mutant recipient, in addition to the expected transformant classes exhibiting UV-resistant (type I) and UV-sensitive (type III) spores, an additional recombinant class was observed (called type II), spores of which exhibited slower germination kinetics following UV irradiation. The results suggested that the spl-1 allele consisted of at least two separable mutations. The DNA region which could rescue the spl-1 allele was localized to a 511-bp region within the spl coding sequence; this region was amplified from the spl-1 mutant chromosome by PCR and sequenced. The region contained two amino acid substitutions, an Arg replacing Gly-168 (G168R) and an Asp replacing Gly-242 (G242D) in the deduced SP lyase sequence, as well as 18 silent mutations. PCR amplification of chromosomal DNA from a selected type II recombinant and sequence analysis of the amplification product confirmed that recombination had indeed occurred between codons 168 and 242 and further localized the point of crossover by using the 18 silent mutations as molecular markers throughout the region. By in vitro mutagenesis, alleles of spl containing all combinations of single and double amino acid substitutions were introduced into the cloned wild-type spl gene. When integrated into the B. subtilis chromosome at the amyE locus, it was observed that although both amino acid substitutions contribute to the spl-1 phenotype, the G168R mutation exerted a much greater effect than did the G242D mutation.

Alleles↗
PubMed2005

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris.

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.

Gram-Positive Endospore-Forming Rods↗
PubMed1986

Biochemistry of fern spore germination: protease activity in ostrich fern spores.

Protease activities were detected in quiescent and germinating spores of the ostrich fern (Matteuccia struthiopteris [L.] Todaro). Peak endopeptidase, aminopeptidase, and carboxypeptidase activities were detected 12 to 24 hours after spores began imbibing under light. There was a correlation between activities of proteases, the onset of a decline in levels of soluble protein, and an increase in levels of free amino acids. The earliest visible event of spore germination, breakage of the spore coat and protrusion of a rhizoid cell, was observed after peak protease activity, 48 to 72 hours after the start of imbibition. Results of this study demonstrate similarities in the pattern of protease activities during germination of ostrich fern spores to those of some seeds.

Journal Article↗
PubMed1998

SASP (small, acid-soluble spore proteins) and spore properties in Bacillus thuringiensis israelensis and Bacillus sphaericus.

Entomopathogenic bacilli B. thuringiensis israelensis (Bti) and B. sphaericus (Bf) exhibit low survival on field application. It was previously shown that their spores are very sensitive to different stress effectors (heat, UV light) and especially to osmotic variations. Since SASP (Small, Acid-Soluble Spore Proteins), alpha/beta and gamma type, are involved in spore tolerance to heat, UV light, peroxide, and salt, they were analyzed in Bti and Bf. The molecular weight, migration pattern and amino acid composition of different SASP were determined and compared with other bacilli, in particular to B. subtilis. A relation between spore osmotolerance, SASP content, and amino acid composition was shown. In addition, the absence of gamma SASP in Bti and Bf is discussed.

Bacillus↗
PubMed2003

Immunocytochemical localization of stachylysin in Stachybotrys chartarum spores and spore-impacted mouse and rat lung tissue.

Stachylysin is a proteinaceous hemolytic agent that is produced by Stachybotrys chartarum. Stachylysin was found, using immunohistochemical and immunocytochemical methods, to be localized in S. chartarum spores/mycelia primarily in the inner wall suggesting that it is constitutively produced. Spores instilled in mouse or rat lung tissues resulted in granuloma formation, which showed the highest stachylysin concentration in the inner wall of the spore and near the spore, with less at distance indicating that it had diffused out from the spore. The in vitro high stachylysin producing strain (58-06) was also highest in vivo, based on immunohistochemistical staining. More stachylysin was observed in the mouse lung tissue at 72 h than at 24 h indicating that production/release is a relatively slow process. The localization of stachylysin in macrophage phagolysosomes suggests that these cells may be involved with hemolysin inactivation. This would be consistent with what is known about asp-hemolysin produced by Aspergillus fumigatus.

Animals↗
PubMed1998

Protein W, a spore-specific protein in Myxococcus xanthus, formation of a large electron-dense particle in a spore.

The gene for the major spore-specific protein, termed protein W, was cloned, and it was found that protein W is composed of 426 amino acid residues including 31% charged (133 residues) and 39% hydrophobic (166 residues) amino acids. In the protein, a motif consisting of five amino acid residues [(V/L/I)-R-E-R-(V/L/I)] is repeated 28 times, and another motif [M-M-(P/G)-Q-G] five times. Protein W is synthesized during a very late stage of development, forming a single, large electron-dense particle (200-400 nm in diameter) inside a spore. X-ray microanalysis of the particle revealed that it contains a high amount of phosphate in addition to calcium and magnesium. It is proposed that protein W consisting of highly charged repetitive sequences is a polyphosphate storage protein to store energy in spores. The disruption of the gene for protein W resulted in delayed fruiting body formation and a lower spore yield.

Amino Acid Sequence↗
PubMed1959

Chemical and morphological studies of bacterial spore formation. III. The effect of 8-azaguanine on spore and parasporal protein formation in Bacillus cereus var. alesti.

The purine analogue, 8-azaguanine, was added to cultures of the parasporal crystal-forming organism Bacillus cereus var. alesti at different times during growth and synchronous sporulation. The effect of its incorporation has been studied with particular reference to cell growth, nucleic acid composition, cytology, and the synthesis of the spore and crystal protein. Additions of the analogue during any stage of growth prevented further cell proliferation and all spore and crystal formation. Since both nucleic acids continued to be formed, cells of an increased size developed, containing large masses of chromatin in the form of condensed balls or axial cords. Lipid-containing inclusions also appeared following these additions and were usually aggregated at the centre or poles of the cells. The analogue could be isolated as the ribonucleotide from both the acid soluble and RNA fractions of these inhibited cells. Additions of the analogue following commencement of sporulation did not prevent either spore or crystal formation or affect the nucleic acid content of the sporulating cells. However, as before, the 8-azaguanine was incorporated into both the acid soluble and RNA of the cells, but not into these fractions of the spores ultimately formed. The implications of these findings are discussed in relation to crystal protein synthesis.

Antimetabolites↗
PubMed2004

Spore morphotypes of Thelohania solenopsae (microsporidia) described microscopically and confirmed by PCR of individual spores microdissected from smears by position ablative laser microbeam microscopy.

Development of Thelohania solenopsae, a parasite of the red imported fire ant (Solenopsis invicta), until recently was thought to include formation of two types of spores: unicellular meiospores, maturing inside sporophorous vesicles in sets of eight (octospores); and Nosema-like binuclear free spores. Megaspores, discovered in 2001, develop primarily in alates and are morphologically distinct from the two previously known types of spores. The role of megaspores in the T. solenopsae life cycle, as well as their existence, has been questioned. The current research includes light and electron microscopic descriptions of the three major spore morphotypes characteristic of T. solenopsae development. In addition, individual octospores and megaspores were isolated into groups of 8-20 from methanol-fixed and Calcofluor-stained smears of the infected ants for subsequent PCR analysis by the laser pressure catapulting function of a position ablative laser microbeam microscope, a technique applied for the first time to research of microsporidia. The PCR-amplified SSU rDNA nucleotide sequences from octospores and megaspores were identical. This, along with the consistency with which megaspores are detected in infected ants, demonstrates that megaspores are integral to the life cycle of T. solenopsae.

Animals↗
PubMed1991

Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA.

Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores.

Bacillus subtilis↗
PubMed2001

Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity.

A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55 degrees C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl(2) indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5' end of sleC. The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.

Amino Acid Sequence↗
PubMed2005

A gene encoding a holin-like protein involved in spore morphogenesis and spore germination in Bacillus subtilis.

We report here studies of expression and functional analysis of a Bacillus subtilis gene, ywcE, which codes for a product with features of a holin. Primer extension analysis of ywcE transcription revealed that a single transcript accumulated from the onset of sporulation onwards, produced from a sigma(A)-type promoter bearing the TG dinucleotide motif of "extended" -10 promoters. No primer extension product was detected in vivo during growth. However, specific runoff products were produced in vitro from the ywcE promoter by purified sigma(A)-containing RNA polymerase (Esigma(A)), and the in vivo and in vitro transcription start sites were identical. These results suggested that utilization of the ywcE promoter by Esigma(A) during growth was subjected to repression. Studies with a lacZ fusion revealed that the transition-state regulator AbrB repressed the transcription of ywcE during growth. This repression was reversed at the onset of sporulation in a Spo0A-dependent manner, but Spo0A did not appear to contribute otherwise to ywcE transcription. We found ywcE to be required for proper spore morphogenesis. Spores of the ywcE mutant showed a reduced outer coat which lacked the characteristic striated pattern, and the outer coat failed to attach to the underlying inner coat. The mutant spores also accumulated reduced levels of dipicolinic acid. ywcE was also found to be important for spore germination.

Bacillus subtilis↗
PubMed2002

Localization of the (1,3)beta-D-glucan synthase catalytic subunit homologue Bgs1p/Cps1p from fission yeast suggests that it is involved in septation, polarized growth, mating, spore wall formation and spore germination.

Schizosaccharomyces pombe Bgs1p/Cps1p has been identified as a putative (1,3)beta-D-glucan synthase (GS) catalytic subunit with a possible function during cytokinesis and polarized growth. To study this possibility, double mutants of cps1-12 and cdc septation mutants were made. The double mutants displayed several hypersensitive phenotypes and altered actin distribution. Epistasis analysis showed mutations prior to septum synthesis were dominant over cps1-12, while cps1-12 was dominant over the end of septation mutant cdc16-116, suggesting Bgs1p is involved in septum cell-wall (1,3)beta-D-glucan synthesis at cytokinesis. We have studied the in vivo physiological localization of Bgs1p in a bgs1delta strain containing a functional GFP-bgs1(+) gene (integrated single copy and expressed under its own promoter). During vegetative growth, Bgs1p always localizes to the growing zones: one or both ends during cell growth and contractile ring and septum during cytokinesis. Bgs1p localization in cdc septation mutants indicates that Bgs1p needs the medial ring and septation initiation network (SIN) proteins to localize properly with the rest of septation components. Bgs1p localization in the actin mutant cps8-188 shows it depends on actin localization. In addition, Bgs1p remains polarized in the mislocalized growing poles and septa of tea1-1 and tea2-1 mutants. During the meiotic process of the life cycle, Bgs1p localizes to the mating projection, to the cell-to-cell contact zone during cell fusion and to the neck area during zygote formation. Also, Bgs1p localization suggests that it collaborates in forespore and spore wall synthesis. During spore germination, Bgs1p localizes first around the spore during isotropic growth, then to the zone of polarized growth and finally, to the medial ring and septum. At the end of spore-cell division, the Bgs1p displacement to the old end occurs only in the new cell. All these data show that Bgs1p is localized to the areas of polarized cell wall growth and so we propose that it might be involved in synthesizing the lineal (1,3)beta-D-glucan of the primary septum, as well as a similar lineal (1,3)beta-D-glucan when other processes of cell wall growth or repair are needed.

Actins↗
PubMed1995

Purification and partial characterization of a spore cortex-lytic enzyme of Clostridium perfringens S40 spores.

A spore cortex-lytic enzyme was purified in an active form from the exudate of fully germinated spores of Clostridium perfringens S40. The enzyme caused attenuation of absorbance in coatless spore suspensions and phase-darkening of the spores, but had minimal activity on isolated peptidoglycan fragments. The enzyme was identified as a 31 kDa protein which is probably an N-acetylmuramyl-L-alanine amidase. The amino-terminal 15 residues of the enzyme were: VLPEPVVPEYIVVHN.

Amino Acid Sequence↗
PubMed2003

Heat resistance of Bacillus spores when adhered to stainless steel and its relationship to spore hydrophobicity.

Twenty-one strains of Bacillus (10 B. stearothermophilus, 3 B. cereus, and 8 B. licheniformis strains) were assayed for spore surface hydrophobicity on the basis of three measures: contact angle measurement (CAM), microbial adhesion to hydrocarbons (MATH), and hydrophobic interaction chromatography (HIC). On the basis of the spore surface characteristics obtained from these assays, along with data on the heat resistance of these spores in water, eight strains of Bacillus (three B. stearothermophilus, three B. cereus, and two B. licheniformis strains) either suspended in water or adhering to stainless steel were exposed to sublethal heat treatments at 90 to 110 degrees C to determine heat resistance (D-value). Significant increases in heat resistance (ranging from 3 to 400%) were observed for the eight strains adhering to stainless steel. No significant correlation was found between these heat resistance increases and spore surface characteristics as determined by the three hydrophobicity assays. There was a significant positive correlation between the hydrophobicity data obtained by the MATH assay and those obtained by the HIC assay, but these data did not correlate with those obtained by the CAM assay.

Bacillus↗
PubMed1977

Studies on the bacterial spore coat 6 effects of alkali extraction on the spore of Bacillus thiaminolyticus.

Thin sections of the spore of Bacillus thiaminolyticus Matsukawa and Misawa show a characteristic surface structure with five ridges, and a series of three district layers. The outer layer of the spore coat was peeled off by SDS sonic treatment, and than the middle layer was solubilized by alkali extraction of the SDS sonic-treated spore. The spores subjected to these treatments were still refractile, heat resistant, and contained dipicolinic acid, but lost their resistance to mechanical shock.

Antigens, Bacterial↗